Immunotoxins:
Immunotoxins (IT's) are chimeric molecules in which cell-binding ligands are coupled to toxins or their subunits. The ligand portion of the immunotoxin is usually a monoclonal antibody (Mab) that binds to selected target cells. The toxin portion of the immunotoxin can be derived form various sources. Most commonly, toxins are derived from plants or bacteria, but toxins of human origin or synthetic toxins (drugs) have been used as well. Toxins used for immunotoxins derived from plants or bacteria all inhibit protein synthesis of eukaryotic cells. Unlike chemotherapeutic molecules, these toxins kill both resting and dividing cells. The toxins share a number of common features: (i) they are synthesized as single chain proteins and are processed either post translationally or in the target cell to which they are delivered into two-chain molecules with interchain disulfide bonds; (ii) the disulfide bond linking the two chains is critical for cytotoxicity; and (iii) all toxins have separate subunits or domains devoded to binding to cells, translocation across membranes, and the destruction of protein synthesis in the target cell. These domains can be separated or genetically manipulated to delete those that are unwanted.
The most widely used plant toxins ricin and abrin, consist of two disulfate-linked polypeptides A and B (Olsnes et al., in Molecular Action of Toxins and Viruses p51-105 (1982)). Another group of plant-derived toxins used in immunotoxins are the ribosome inactivating proteins (RIPs). These molecules are single-chain proteins frequently found in plants and have similar enzymatic properties as the A-chain of ricin (reviewed in Stirpe and Barbieri FEBS 195:1 (1986)). The cross-linker used to join the Mab and the toxin must remain stable extracellularly, but labile intracellularly so that the toxin fragment can be released in the cytosol. The choice of cross-linker depends on whether intact toxins, A-chains or RIPs are used. A-chains and RIPs are generally coupled to the Mab using linkers that introduce a disulfide bond between the ligand and the A-chain (Myers et al., J. Immunol. Meth. 136:221 (1991)). Bonds that cannot be reduced render these immunotoxins much less toxic or nontoxic, probably because the A-chain must be released from the ligand by reduction to be cytotoxic. Intact toxins are usually linked to ligands using non-reducible linkages (such as thioether) to prevent release of the active free toxin in vivo.
RIPs, efficiently inhibit eukaryotic protein synthesis. Gelonin is a type I RIP (single catalytic chain), which has an advantage above type II RIPs in that type II RIPs have in addition to the catalytic chain, a cell-binding lectin-like B-chain. Because gelonin has no cell-binding lectin-like B-chain, it is unable to bind to cell membranes in the absence of a targeting agent and therefore has a low nonspecific toxicity. Even in comparison with another type I RIP (saporin), LD50 studies in mice have shown that native gelonin is approximately 10-fold less toxic than saporin, and thus may be particularly suitable for therapeutic applications. Moreover, immuno-conjugates with gelonin when targeted to cells have low IC50 values, inhibit a greater percentage of target cells and require less exposure time in comparison to other toxins. For these reasons, the low native toxicity and the high specific toxicity, the therapeutic window is very high for gelonin. Gelonin is among the most promising toxins used for the construction of ITs. In direct comparison experiments, gelonin was superior to two of the most popular toxins, ricin A chain and Pseudomonas exotoxin A (Fishwild et al Clin Exp Immunol 97:10 (1994)). The cDNA of gelonin was recently isolated (Better et al J Biol Chem 270:14,951 (1995)), allowing the construction of single chain antibody-toxin fusion proteins (ScFv-IT). A complete Mab consists of two complete heavy and two complete light chains and has a molecular weight of 150 kDa. An immunotoxin molecule based on a whole antibody will have a molecular weight in the range of 200 kDa, depending on the type of toxin and the amount of toxin molecules coupled per antibody. A single chain antibody fragment (ScFv) however, consists of only the variable part of the heavy and light chain coupled via a short linker and has a molecular weight of approximately 25 kDa. When a toxin molecule is directly fused to a ScFv molecule by genetic engineering, the size of the ScFv-immunotoxin molecule thus obtained, will be a factor 4 smaller when compared to a complete antibody-immunotoxin molecule. Since tumor penetration is mainly dependent on size (the smaller the IT the better the tumor penetration), it is prefered to use ScFv-IT molecules. In addition, the serum half-live of a ScFv-IT is much shorter when compared to a complete antibody-immunotoxin molecule, thus reducing the non-specific systemic toxicity.
CD80/CD86 costimulatory molecules:
CD80 (B7.1) is a monomeric transmembrane glycoprotein with an apparent molecular mass of 45-65 kDa and is a member of the immunoglobulin superfamily (Freeman et al. J. Immunol. 143:2714, (1989)). It was initially reported that the expression of the CD80 molecule was restricted to activated B cells (Freeman et al., J. Immunol. 143:2714, (1989)) and monocytes stimulated with IFN-.gamma. (Freedman et. al., Cell. Immunol. 137:429, (1991)). More recently, CD80 expression has also been found on cultured peripheral blood dendritic cells (Young et al. J. Clin. Invest. 90: 229 (1992)). The expression of the CD80 molecule in a number of normal and pathological tissues has been examined by immunohistochemistry using an anti-CD80 monoclonal antibody (Vandenberghe et. al., Int. Immunology 5:317 (1993)). In addition to the staining of activated B cells, it was shown that the CD80 molecule is constitutively expressed in vivo on dendritic cells in both lymphoid and non-lymphoid tissue. Monocytes/macrophages were only found to be positive under inflammatory conditions and endothelial cells were always negative. Interestingly, the number of CD80 positive cells in skin lesions of patients with acute GVHD was strongly increased when compared to normal skin. This expression pattern of CD80 on different antigen presenting cells (APCs), strongly suggests an important costimulatory role in T-cell activation.
It has recently been demonstrated that CD80 is a member of a family of closely related molecules molecules, that can functionally interact with CD28 (Hathcock et al. Science 262:905 (1993); Freeman et al. Science 262:907 (1993); Azuma et al. Nature 366:76 (1993)). The second member of this family, B7.2 or CD86, is also a transmembrane glycoprotein, with an apparent molecular mass of approximately 70 KDa and is also a member of the immunoglobulin superfamily (Freeman et al. Science 262:907 (1993); Azuma et al. Nature 366:76 (1993)). The CD86 molecule seems to have a very similar distribution pattern as CD80, with the exception that induction of cell-surface expression seems to be faster and that it is present on freshly isolated monocytes.
Transplant Rejection:
Incompatibility for the histocompatibility antigens, both major (MHC) and minor antigens, is the cause for graft rejection. Both CD4+ helper T cells (Th) and CD8+ cytotoxic T cells (CTL) are involved in the rejection process. Activation of T cells after transplantation is the result of ligand-receptor interactions, when the TcR/CD3 complex recognizes its specific alloantigen in the context of the appropriate MHC molecule. To induce proliferation and maturation into effector cells, T cells need a second signal in addition to the one mediated by the TcR/CD3 complex. Intercellular signaling after TcR/MHC-peptide interaction in the absence of the costimulatory signal results in T-cell inactivation in the form of clonal anergy (Mueller et al. Annu. Rev. Immunol. 7:445 (1989)). It has been demonstrated that blocking CD80/CD86, when combined with a donor-specific cell transfusion, can prevent the rejection of MHC-mismatched cardiac allografts in a rat model (Lin et al. J. Exp. Med. 178:1801 (1993)). In addition, it has been demonstrated that co-stimulation of T cells via the cross-linking CD28 is resistant to the inhibitory activity of the immunosuppressive drug cyclosporin A (June et al. Immunol. Today 11:211 (1990)). This demonstrates the importance of the CD80/CD86-CD28 interaction in the rejection of transplants. It has been suggested and demonstrated in rodent models, that blocking both CD80 and CD86, thereby preventing the ligation of CD28 on T cells, can prevent rejection of allo-transplants. However, no prior art exists that an immunotoxin targeting CD80 or CD86 can prevent alloantigen-specific T cell activation and thus allo-graft rejection.
Autoimmune diseases:
A number of studies indicate that costimulation through CD28 ligation might be the initiating event in autoimmunity. The potential of both a primary signal via the TcR and CD80 as a costimulatory signal for the generation of autoimmune diabetes has clearly been proven with transgenic mice (Guerder et al., Immunity 1:155 (1994); Harlan, et al., PNAS 91:3137 (1994)). In these studies, it is hypothesized that tolerance to peripheral antigens is induced by triggering the TcR in the absence of essential costimulatory signals. Mice expressing both CD80 and a high level of primary antigens (MHC molecules or viral glycoproteins) on pancreatic beta cells developed autoimmune diabetes. The critical role of the absence of CD80-mediated costimulation in the induction and maintenance of tolerance to peripheral antigens, and of the CD80-mediated signalling in the breakdown of T-cell nonresponsiveness, causing autoimmunity, was obvious.
The role of the CD80/CD86-CD28 interaction in the chronic activation state of T cells, which have been implicated in autoimmune diseases, has been strongly suggested in various studies. Using immunohistochemical techniques, strong CD80 expression has been found in lesions of autoimmune diseases, such as rheumatoid arthritis and psoriasis. Furthermore, it has been demonstrated that blocking CD80/CD86-CD28 interaction could block autoantibody production and prolongation of life in a murine model of autoimmune disease that closely resembles systemic lupus erythematosus in humans (Finck et al., Science 265:1225 (1994)).
Hodgkin's Disease:
Hodgkin's Disease (HD) comprises a group of malignant lymphomas with common clinical and pathologic features. The diagnosis is based on a disrupted lymph node architecture and the presence of the presumed malignant mononucleated Hodgkin and the multinucleated Reed-Stemberg (H-RS) cells in the right setting, consisting mainly of small lymphocytes, and a variable admixture of histiocytes, eosinophils and plasma cells. The etiology of HD and the origin of the H-RS cells remains unclear. Four histologic subtypes are recognized: Lymphocytic predominance (5-10%), nodular sclerosis (40-70%), mixed cellularity (20-40%) and lymphocytic depletion (5%). Prognosis is mainly determined by the stage of the disease as determined according to Ann-Arbor classification. An unbalanced production of cytokines in active HD has been associated with constitutional "B" symptoms as fever, night sweats, generalized itching and weight loss.
Immunohistology of HD lymph nodes shows that the majority of T cells surrounding the H-RS cells are activated (IL-2R+, CD40L+) CD4+ memory T cells. H-RS cells express strongly CD30, CD40, IL-2R, CD80, CD86, CD71 (Transferrin Receptor) and adhesion molecules such as ICAM-1. Cytokines produced by H-RS cell lines include IL-6, IL-8, TNF-alpha, TNF-beta or lymphotoxin, GM-CSF, IL-1, IL-3, IL-10 and TGF-beta. These cytokines are very likely responsible for the clinical features of HD like eosinophilia, "B" symptoms, acute phase reactants, thrombocytosis and sclerosis of HD involved tissues. HD is a tumor highly responsive to both chemotherapy and radiotherapy. Most patients with early stage disease can be cured with single modality treatment. The majority of patients presenting with advanced disease can also achieve complete remission. However a significant proportion (about 40%) of patients will have recurrence of their disease. For patients not attaining a complete remission on first and second line chemotherapy initially or at relapse the outcome is dismal. Although responses to salvage regimens are present, long term disease-free survival is unusual with only 20% 5 year survival. In patients relapsing after chemotherapy intensive chemotherapy followed by autologous bone marrow transplantation seems to be a good option in those with a sensitive relapse, but long term results have to be awaited. A further concern are secondary tumors and heart disease in patients cured from HD, probably due to toxicity of the chemo and/or radiotherapy. Immunotherapy may be a good alternative initially in HD patients with primary resistant disease or relapse, and if successful probably also in patients with earlier stages of HD.
Problems posed in the present invention
As is reviewed above, it is known that certain surface molecules are upregulated or overexpressed in diseases of the immune system. A specific example of such molecules are CD80 and CD86 present on antigen presenting cells (APCs). A number of these upregulated surface antigens have been proven to be involved in T cell-activation. For the treatment of diseases of the immune system which involve these surface antigens, researchers have focused on ways to block these surface antigen molecules (e.g. by using Mab directed to these surface antigen molecules) so that they cannot function normally because they cannot transmit the necessary signal to the T-cell.
The problem posed in the present invention may be formulated as providing an alternative method for treating or preventing diseases of the immune system, more particularly for treating or preventing allograft rejection, autoimmune diseases and various malignancies of lymphoid origin.
To solve this problem the present inventors have been able to prove that immunotoxins can, surprisingly, also be used in the field of treating diseases of the immune system, more particularly for treating allograft rejection, autoimmune diseases and malignancies of lymphoid origin such as Hodgkin's disease. It should be stressed that the use of immunotoxins is well known in the field of treating tumors. Surprisingly, however, the present inventors could show that the technology of immunotoxins could also be applied in a way that it indirectly influences the activation state of T-cells, implying that the inhibition of protein synthesis in one cell type, the antigen presenting cell, has an effect on a second cell type, the antigen-specific T-cell. This is surprising, since protein expression for essential costimulatory molecules such as CD80 and CD86 on the antigen-presenting cells is not immediately eliminated. In other words, only de novo synthesized CD80/CD86 molecules appear to be essential to activate T cells and not the CD80/CD86 molecules which were present before, or immediately after, the addition of immunotoxins.
This alternative method for blocking the interaction between APC's and T cells based on the usage of CD80/CD86 immunotoxins shows a considerable advantage over the existing methods using anti-CD80/86 Mab's in a way that killing the APC's, instead of only blocking the CD80/86 molecules, also inhibits signalling from other accessory molecules (such as LFA-1, LFA-3, ICAM or others) on the APC's which are known, in addition to CD80 and CD86, to be involved in T cell triggering (van Gool et al. Res. Immunol. 146:183 (1995)). Furthermore, it is also known that both CD80 and CD86 are important in T cell triggering by APC's (van Gool et al. Res. Immunol. 146:183 (1995) which implies that both molecules need to be blocked to efficiently inhibit T cell activation. The approach using Mab's would therefore involve the usage of at least two different Mab's (anti-CD80 and anti-CD86) whereas only one immunotoxin (CD80-IT or CD86-IT) can be used following the alternative method of the present invention.
It is further known that radio-immunoconjugates and immunotoxins are largely unexplored in Hodgkin's Disease. Only preliminary studies have been reported. Yttrium-labeled anti-ferritin and CD30-saporin have shown promising results in phase I-II studies in end stage patients with HD. Radio-immuno-therapy has the disadvantage of considerable hematologic toxicity, difficulties with dosing and the fact that HD patients have already received significant amounts of radiation. The CD30-immunotoxin is a much more focussed approach directed to Hodgkin/Reed-Sternberg (H-RS) cells. One small trial is reported using ITs in HD (Falini et al Lancet 339:1195 (1992)). In this trial, 4 chemotherapy resistant patients were treated with CD30-saporin bolus infusions (0.6 mg/kg I.V. in one or two doses). No severe toxicity was observed. Patients suffered from minor episodes of fever, malaise, anorexia, minor liver function disturbances and thrombocytopenia with no Vasicular Leak Syndrome (VLS) or hypo-albuminemia. No maximum tolerated dose was established. While toxicity was limited, 3/4 patients achieved a transient remission and a minimal response. CD30 may not be an ideal antigen for an immunotoxin in HD. This is because soluble CD30 is present in the blood of patients with advanced HD (Gause, et al., Blood 77:1983 (1991)) and this antigen is also expressed on activated T cells, which are present in large numbers in HD. Elimination of these cells by a CD30-immunotoxin may aggravate the already existing T cell function defects in HD and can cause a T cell cytokine release syndrome.
Target antigens for potential ITs, strongly expressed on H-RS cells are as indicated above CD30, CD40, IL-2 receptor (CD25), CD80, CD86 and the Transferrin-Receptor (CD71). An anti-CD80-IT or anti-CD86-IT is clearly the best, because of the anticipated side effects of the others. CD71 is a poor target antigen because it is expressed on all rapidly dividing cells. Bone marrow toxicity is a significant problem with an anti-CD71-IT (See example 6 below). Anti-CD40-IT will eliminate all (resting and activated) B cells and activated endothelial cells. In addition, anti-CD40-IT generate monokine release and anti-CD30-IT and anti-CD25IT will augment T lymphokine release. Furthermore, soluble forms of CD25, CD30 and CD40, present in HD patients, complicate dosing and reduce efficacy. Using anti-CD80-IT and anti-CD86IT might result in the elimination of activated dendritic cells. This will be only transient, as these cells will be rapidly replaced from stem cells. As a matter of fact, elimination of the dendritic cells by anti-CD80-IT and anti-CD86-IT even has a positive effect, since antigen presentation is blocked and therefore the capacity of the patient to produce antibodies to the immunotoxin response is blocked. The CD80 and CD86 antigens are thus likely the best antigens for several reasons: (i) antigen heterogeneity, most HD tumors express CD80 and/or CD86; (ii) soluble forms of CD25, CD30 and CD40 complicate dosing and reduce efficacy; (iii) antigen modulation (i.e. cleavage from the cell membrane by proteases) of molecules such as CD25, CD30 and CD40 negatively influences uptake of IT's into the cells; (iv) there will be no antibody response to anti-CD80IT and anti-CD86-IT.
In this regard, the problem posed in the present invention may be regarded as chosing suitable surface molecules which would direct immunotoxins to the malignant types of cells present in patients suffering from Hodgkin's Disease.
To solve this problem the present inventors have been able to prove that surprisingly CD80 and/or CD86 can be used to design anti-CD80 toxin conjugates and/or anti-CD86 toxin conjugates which can effectively be used in vitro to inhibit the growth of tumor cell lines (example 6). From this finding it could be concluded that such immunotoxins can also be used to treat Hodgkin's disease in vivo.
In short, the present invention aims particularly at providing new immunotoxins or compositions comprising the same, methods for preparing the same and methods for treating diseases of the immune system involving the use of these immunotoxins or compositions.
The invention also aims at providing a medicament comprising the same as well as a method for preparing said medicament.
In particular, the present invention aims at methods for preventing allograft rejection.
More particularly, the present invention aims at methods for treating autoimmune diseases.
Also, the present invention aims at providing methods for treating various malignancies of lymphoid origin, more particularly Hodgkin's disease.
Also, the present invention aims at providing donor organs and isolated lymphocytes incubated ex vivo with said immunotoxins or said compositions, as well as a method for their preparation.
All the aims of the present invention are considered to have been met by the embodiments as set out below.